This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Proteins must fold correctly in order to attain biological function. Concurrently, protein aggregation and misfolding are key contributors to many devastating human diseases such as Alzheimer's disease and prion-mediated infections. Unlike other more conventional molecular chaperones, the caseinolytic protease B (ClpB) and its yeast homolog heat-shock protein 104 (Hsp104) have the remarkable ability to rescue proteins from a previously aggregated state. Members of the ClpB/Hsp104 family form hexameric ring structures of ~600 kDa in molecular weight and convert chemical energy derived from ATP-binding and [unreadable]hydrolysis into mechanical work. The goal of this research is to provide a detailed mechanistic understanding how ClpB and Hsp104 facilitate the dissociation of previously aggregated proteins. Project 1: Structure of the ClpB hexamer ClpB is an ATP-dependent molecular machine, which forms a 600-kDa hexameric ring structure and belongs to the Clp/Hsp100 family of AAA+ proteins. Unlike other molecular chaperones, however, ClpB neither promotes the forward folding nor prevents the aggregation of proteins. Instead, ClpB has the remarkable ability to rescue stress-damaged proteins from an aggregated state;a function that is essential for induced thermotolerance. While the structure-function relationship is beginning to be understood, it remains unclear how ClpB converts the energy derived from ATP binding and hydrolysis into mechanical work. Project 2: Structure of the Hsp104 molecular chaperone First discovered as an essential component in the yeast stress response, Hsp104 is an ATP-dependent protein-remodeling factor, which, like ClpB, can rescue stress-damaged proteins from a previously aggregated state. While the ability to do so is shared with bacterial ClpB, the underlying mechanism is different. For instance, it has been proposed that the second AAA domain of Hsp104 is essential for oligomerization and is required for substrate binding while the reverse is true for ClpB. To provide a detailed mechanistic understanding of Hsp104, we are using cryoEM to determine the structure of Hsp104 both in the nucleotide-bound and -free states